Dataset Information

C18 nanoLC-MS and Glycogenomics to Facilitate O-Glycan profiling in Human Cells

ABSTRACT: O-glycosylation is an abundant modification throughout the human proteome and plays a role in, amongst others, regulation of protein cleavage, protein folding, and modulation of cell-cell and -matrix interactions. O-glycosylation type and structure are important for biological function, demanding analytical methodologies that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the non-specialist and/or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the non-reductive release of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nano-liquid chromatography coupled to Orbitrap mass spectrometry (nanoLC-MS/MS). The implementation of glycogenetically engineered cell material proved a way into the complex structural annotation of O-glycan types and isomers. Using human keratinocytes as model system, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc and O-GalNAc glycosylation, with the latter carrying both elongate Core 1 and Core 2 structures and varying numbers of fucoses and sialic acids. The method, including the now well characterized standards, provide the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Keratinocyte, Skin, Kidney

SUBMITTER: Noortje de Haan

LAB HEAD: Hans Wandall

PROVIDER: PXD029644 | Pride | 2022-04-04

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	210818_Lumos__LC1200__nanoFlex_NH21036_01_01_Ogly.raw	Raw
	210818_Lumos__LC1200__nanoFlex_NH21036_01_02_Ogly.raw	Raw
	210818_Lumos__LC1200__nanoFlex_NH21036_01_03_Ogly.raw	Raw
	210818_Lumos__LC1200__nanoFlex_NH21036_01_04_Ogly.raw	Raw
	210818_Lumos__LC1200__nanoFlex_NH21036_01_05_Ogly.raw	Raw

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Publications

In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.

de Haan Noortje N Narimatsu Yoshiki Y Koed Møller Aasted Mikkel M Larsen Ida S B ISB Marinova Irina N IN Dabelsteen Sally S Vakhrushev Sergey Y SY Wandall Hans H HH

Analytical chemistry 20220304 10

O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth pro ...[more]

PMID: 35245040

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Project description:The majority of cell surface and secreted proteins are modified by glycans and this glycosylation process plays an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signalling. Glycosylation plays an important role in myogenesis and the development of muscle tissue but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during in vitro myogenesis. We observed wide-spread changes in the abundance of several receptors and important enzymes regulating glycosylation. Quantification of released N-linked glycans via glycomic analysis confirmed remodeling of the glycome with a switch in sialic acid linkages, and changes in di-galactosylation and paucimannosylation. Quantitative glycoproteomic analysis with stable isotope labelling, enrichment of glycopeptides and analysis via multiple fragmentation approaches identified precise glycoproteins containing these regulated glycans including integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins including the up-regulation of Galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early defect in the myogenic program. We also observed similar changes in N-glycosylation and the up-regulation of LGALS1 during postnatal skeletal muscle development in mice. Finally, treatment of new-born mice with recombinant adeno-associated viruses to express LGALS1 in the musculature resulted in enhanced muscle mass. Our data will be a valuable resource to further understand the role of glycosylation and lectins on myogenesis and may aid in the development of intervention strategies aimed at promoting healthy muscle.

Project description:Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between a2,3- and a2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.

Project description:Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). CRC is known to display glycosylation alterations. Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer, such as cell signaling and adhesion, and may can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples from patients with tumors in the right or left colon and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left side of the colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways, regardless of tumor location.

Dataset Information

C18 nanoLC-MS and Glycogenomics to Facilitate O-Glycan profiling in Human Cells

Dataset's files

Publications

In-Depth Profiling of <i>O</i>-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.

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